PROJECT ABSTRACT Aberrant transcription in the human genome can dramatically disrupt development and both induce and worsen diseases like cancer. In Triple Negative Breast Cancer (TNBC), dysregulated transcription manifests in chronic pathway activation, such as inflammatory signaling mediated by the Nuclear Factor-kappaB (NF-?B) family of transcription factors, as well as global alterations in gene activation or suppression through epigenetic complex dysfunction. EZH2 is the catalytic component of Polycomb Repressive Complex 2 (PRC2), an epigenetic complex responsible for trimethylation of lysine 27 of histone H3 (H3K27me3), which promotes chromatin condensation and gene silencing. EZH2 overexpression and hypermethylation is implicated in multiple cancers including TNBC, but paradoxical non-canonical functions of EZH2 are increasingly reported. Our laboratory has demonstrated that EZH2 is a transcriptional activator of the gene encoding NF-?B transcription factor RelB, and supports tumor initiating cell growth in TNBC cells. Preliminary findings further suggest EZH2 colocalizes with NF-?B transcription factor RelA and transcriptionally activates NF-?B target genes. We predict a novel EZH2-NF-?B signaling axis contributes to TNBC development and progression, and we propose to elucidate the mechanism and cancer-related implications of this signaling using TNBC cell lines and patient- derived organoids. We hypothesize that EZH2 functions as a PRC2-independent transcriptional activator in TNBC functioning cooperatively with the NF-?B factor RelA. In Aim 1, we will define the non-canonical EZH2 gene activation signature in TNBC cells by determining global EZH2 localization and EZH2-dependent gene expression including NF-?B target genes, and further determining which activated genes are PRC2-independent. In Aim 2, we will characterize the EZH2 and NF-?B relationship in TNBC cells by determining which genes are co-occupied and co-regulated by EZH2 and RelA, and whether EZH2 and RelA interact directly or functionally. In Aim 3, we will elucidate mechanisms of EZH2 non-canonical function in TNBC cells by determining whether EZH2 overexpression and/or post-translational modification are sufficient and necessary to drive non-canonical EZH2 activity in relation to NF-?B. The proposed research will take place in the collaborative and productive research environment at the University of North Carolina at Chapel Hill in the laboratory of Dr. Albert Baldwin, a longstanding expert in the field of NF-?B signaling and its role in cancer, with the support of co-sponsor Dr. Brian Strahl, a recognized expert in epigenetics and chromatin biology. Completion of these studies will define and determine the contributions of non-canonical EZH2-NF-?B signaling in TNBC cells. This will fundamentally advance breast cancer and epigenetics biology, and illuminate both pitfalls in the current EZH2-targeting strategy and novel targets to consider in developing and advancing therapeutics for TNBC.